Respiratory Syncytial Virus (RSV), a negative strand virus of the paramyxoviridae family, is a major cause of lower pulmonary tract disease, particularly in young children and infants. The parenteral administration of formalin-inactivated RSV (FI-RSV) as a vaccine has been associated with enhanced disease in RSV-naive recipients (seronegative) who subsequently became infected with wild-type RSV. The enhanced disease was characterized by an increased proportion of eosinophils in both the peripheral blood and lungs of affected individuals (Kim et al., Am. J. Epidemiol. 89:422-434 (1969); Kim et al., Pediatric Res. 10:75-78 (1976)). Recent studies in rodents have indicated that FI-RSV induces a T-helper 2 (TH2) immune response, whereas live attenuated viral vaccine are preferentially associated with T-helper 1 (TH 1) responses.
RSV contains two prominent outer envelope glycoproteins, fusion (F) protein and attachment (G) protein, that are important for viral infectivity and thus serve as reasonable targets for the design of a subunit vaccine to RSV. It has previously been shown that the generation of neutralizing antibodies to RSV by an F-protein-based vaccine can be greatly increased by the inclusion of G protein (Hancock et al., J. Virol. 70:7783-7791 (1996)). However, in attempting to understand the molecular basis for FI-RSV-induced enhanced disease, it has previously been shown that the native attachment (G) glycoprotein of RSV is sufficient to prime for atypical pulmonary inflammation characterized by pulmonary eosinophilia associated with high production levels of Interleukin-5 (IL-5), a TH2 cytokine (Hancock et al., J. Virol. 70:7783-7791 (1996)). In fact, the in vivo depletion of IL-5 significantly reduces the eosinophilic response in bronchoalveolar lavage cells of G protein-immunized mice challenged with RSV. The response to G protein was shown to be T cell mediated by transfer of G protein-specific CD4+ T cell lines into naive recipient mice, resulting in atypical pulmonary inflammatory responses upon subsequent challenge (Alwan et al., J. Exp. Med. 179:81-89 (1994)).
The immune responses elicited by native RSV G protein and a series of overlapping peptides (shown in FIG. 2) extending from amino acids 48 to 294 of G protein have been characterized as described herein. In stimulation assays of splenocytes from G protein-vaccinated mice, one peptide (19, spanning amino acids 184-198) was dominant in its ability to stimulate spleen cell proliferation in BALB/c mice (FIG. 3). In these mice, in the absence of any similar effect from other G protein-derived peptides, the use of peptide 19 as an antigen resulted in a stimulation of spleen cell proliferation that was 15-fold above background levels. Peptide 19 was also found to be the major region of the G protein involved in cytokine release, as both IFN-xcex3 and IL-5 were detected in the induction of supernatants from cultures of splenocytes derived from G protein-vaccinated BALB/c mice (FIGS. 4A and 4B). Peptide 19 (amino acids 184-198 of the RSV G protein) specifically induces pulmonary eosinophilia in BALB/c mice. Mice vaccinated with peptide 19 conjugated to keyhole limpet hemocyanin (KLH) showed significant pulmonary eosinophilia (39.5% of total bronchoalveolar lavage cells) upon subsequent challenge with live RSV (FIG. 5). In contrast, mice immunized with a peptide containing amino acids 208-222 (peptide 22) conjugated to KLH exhibited minimal pulmonary eosinophilia (3.3%). Mutations in the amino acid sequence of peptide 19 abrogated the ability to predispose mice for pulmonary eosinophilia (FIG. 6). Furthermore, in additional mouse strains utilized as described herein, peptides 15 (amino acids 159-174), 17 (amino acids 171-187) and 18 (amino acids 176-190) also showed significant ability to induce proliferative responses of G protein-primed splenocytes (FIGS. 11A-11C).
The in vivo depletion of CD4+ cells abrogated pulmonary eosinophilia in mice vaccinated with the peptide 19 conjugate, whereas the depletion of CD8+ cells had a negligible effect (FIG. 8). These data indicate an association between peptide 19 of RSV G protein and the CD4+ T cell-mediated induction of pulmonary eosinophilia in response to live RSV challenge, suggesting that peptide 19-specific CD4+ T cells are the causative agent of pulmonary eosinophilia. In analyzing human peripheral blood cells from 43 donors, 6 showed reactivity to RSV G protein, 3 of which responded to peptide 19 (FIG. 7). This data suggests that peptide 19 may be involved in the onset of bronchiolitis, atopy or asthma that is sometimes observed following RSV infection of seronegative infants (Welliver and Welliver, Pediatrics in Review 14:134-139 (1993)). Taken together, these data indicate that the region of RSV G protein spanning amino acids 159-198 has the capacity to prime vertebrates for pulmonary eosinophilia and thus may mediate enhanced disease upon subsequent infection of the vertebrate with RSV.
Accordingly, the invention pertains to an altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with RSV. In a particular embodiment, the enhanced disease is atypical pulmonary inflammation, particularly pulmonary eosinophilia. In one embodiment, the alteration is in the region from amino acid 184 to amino acid 198 of the RSV G protein. In another embodiment, the alteration is in a region selected from the group consisting of the region from amino acid 159 to amino acid 198 of the RSV G protein, the region from amino acid 159 to amino acid 174 of the RSV G protein, the region from amino acid 171 to amino acid 187 of the RSV G protein, and the region from amino acid 176 to amino acid 190 of the RSV G protein. In an alternate embodiment, the alteration results in inhibition of priming for IL-5 secretion by the altered G protein or polypeptide relative to wild type G protein. In another embodiment, the alteration results in enhancement of priming for IFN-xcex3 secretion by the altered G protein or polypeptide relative to wild type G protein.
The invention also pertains to a nucleic acid molecule encoding an altered G protein or polypeptide of RSV, where the altered protein or polypeptide retains immunogenicity and, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV. In one embodiment, the alteration is in the region from amino acid 184 to amino acid 198 of the RSV G protein. In another embodiment, the alteration is in a region selected from the group consisting of the region from amino acid 159 to amino acid 198 of the RSV G protein, the region from amino acid 159 to amino acid 174 of the RSV G protein, the region from amino acid 171 to amino acid 187 of the RSV G protein, and the region from amino acid 176 to amino acid 190 of the RSV G protein.
The invention also encompasses DNA constructs comprising a nucleic acid molecule described herein operably linked to a regulatory sequence. In a particular embodiment, the invention pertains to a chimeric DNA construct comprising: (a) a nucleic acid molecule encoding an altered G protein or polypeptide of RSV, where the altered protein or polypeptide retains immunogenicity and, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV; (b) a nucleic acid molecule encoding all or an immunogenic portion of F protein of RSV; and (c) a regulatory sequence operably linked to both the F and altered G proteins.
The invention also relates to a recombinant host cell comprising a DNA construct described herein, as well as to a method of producing an altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV, comprising maintaining a recombinant host cell of the invention under conditions suitable for expression of the altered G protein or polypeptide.
The invention also pertains to a method of producing a chimeric polypeptide comprising an altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV, and all or an immunogenic portion of F protein of RSV.
The invention also pertains to the use of the altered G protein or polypeptide, or recombinant host cell for expression thereof, for the manufacture of a medicament, such as a vaccine.
The invention further relates to an immunogenic composition comprising a physiologically acceptable medium and an altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into an immunogenic composition and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV. In a particular embodiment, the immunogenic composition results in inhibition of priming for IL-5 secretion relative to an immunogenic composition comprising wild type G protein. In another embodiment, the immunogenic composition results in enhancement of priming for IFN-xcex3 secretion relative to an immunogenic composition comprising wild type G protein. In one embodiment, the alteration is in the region from amino acid 184 to amino acid 198 of the RSV G protein. In another embodiment, the alteration is in a region selected from the group consisting of the region from amino acid 159 to amino acid 198 of the RSV G protein, the region from amino acid 159 to amino acid 174 of the RSV G protein, the region from amino acid 171 to amino acid 187 of the RSV G protein, and the region from amino acid 176 to amino acid 190 of the RSV G protein. The immunogenic composition can also comprise all or a portion of RSV F protein.
The invention also pertains to a vaccine composition comprising an immunologically effective amount of altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into a vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with RSV. In one embodiment, the alteration is in the region from amino acid 184 to amino acid 198. In another embodiment, the alteration is in a region selected from the group consisting of the region from amino acid 159 to amino acid 198 of the RSV G protein, the region from amino acid 159 to amino acid 174 of the RSV G protein, the region from amino acid 171 to amino acid 187 of the RSV G protein, and the region from amino acid 176 to amino acid 190 of the RSV G protein. The vaccine composition can also comprise an immunologically effective amount of all or a portion of RSV F protein. In particular embodiments, the vaccine compositions further comprise an adjuvant.
The invention further relates to a method of inhibiting induction of enhanced disease after vaccination and subsequent infection of a vertebrate with RSV, comprising administering an altered RSV G protein or polypeptide, where said altered G protein or polypeptide retains immunogenicity and, when incorporated into a vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with RSV.
The invention also relates to a vaccine comprising a physiologically acceptable vehicle and an effective amount of a nucleic acid molecule encoding an altered G protein or polypeptide of RSV, where said altered G protein or polypeptide retains immunogenicity and, when incorporated into a vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with RSV. In one embodiment, the vaccine further comprises a transfection-facilitating agent.
The invention also relates to a method of inducing an immune response in a vertebrate, comprising administering to said vertebrate an amount of DNA encoding an altered RSV G protein or polypeptide effective to induce an immune response, optionally with a transfection-facilitating agent, where said altered G protein or polypeptide retains immunogenicity and, when incorporated into a vaccine and administered to a vertebrate, provides protection without inducing enhanced disease upon subsequent infection of the vertebrate with RSV.
The invention also relates to a method of immunizing a vertebrate against RSV, comprising administering to the vertebrate a composition comprising an immunologically effective amount of altered G protein or polypeptide of RSV which retains immunogenicity and which, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV.
The invention also pertains to a method of immunizing a vertebrate against RSV, comprising administering to the vertebrate a composition comprising an immunologically effective amount of a nucleic acid molecule encoding an altered G protein or polypeptide of RSV, where said altered G protein or polypeptide retains immunogenicity and, when incorporated into an immunogenic composition or vaccine and administered to a vertebrate, does not induce enhanced disease upon subsequent infection of the vertebrate with RSV.
In one embodiment, the composition further comprises an immunologically effective amount of all or a portion of RSV F protein, or a nucleic acid molecule encoding an immunologically effective amount of all or a portion of RSV F protein, respectively. In another embodiment, the vertebrate is an RSV seronegative human.